Inhibition Of Oocyte Maturation Research Articles

O-077 Depletion of MFF from oocytes impairs mitochondrial dynamics, leading to inhibited oocyte maturation and early embryonic development in mice

Abstract Study question To investigate the effect of mitochondrial fission factor (MFF) on oocyte competence and female fertility using an oocyte-specific Mff knockout mouse model. Summary answer Oocyte-specific Mff deficiency hindered oocyte maturation and early embryonic development via regulation of mitochondrial dynamics, resulting in female subfertility. What is known already Emerging studies have demonstrated that mitochondrial dynamics play a vital role in oocyte maturation and early embryo development. MFF is one of the crucial proteins regulating mitochondrial dynamics. Our previous studies have indicated that Mff is necessary for oocyte competence and fertility using a global Mff knockout mouse model. However, further investigation is needed to determine whether the deletion of Mff in oocytes could affect oocyte competence and early embryonic development by regulating mitochondrial dynamics. Study design, size, duration The oocyte-specific Mff knockout (Mfffl/fl; Gdf9-Cre) mice were generated using the Cre-LoxP conditional knockout system. The 2-month-old female mice from each group (Mfffl/fl; Gdf9-Cre or Mfffl/fl, n = 6) were mated to assess the fecundity over 10 months. The maturation and mitochondrial dynamics of germinal vesicle (GV) oocytes were determined. The spindle and chromosome morphologies of ovulated oocytes were also analyzed. Additionally, the 2-cell embryos were collected and cultured in vitro to assess early embryonic development. Participants/materials, setting, methods Female mice from each group were mated with adult males to assess the fecundity. GV oocytes, ovulated oocytes, and 2-cell embryos were collected and cultured in vitro after superovulation, as indicated. Mitochondria, spindle, and chromosome were stained and then imaged using confocal microscopy. The microscopic morphology of mitochondria was assessed using electron microscopy. Main results and the role of chance The Mfffl/fl; Gdf9-Cre mice consistently exhibited reduced fertility with a significant decrease in litter size (3.00 vs. 5.42, p < 0.001), number of litters per female (4.33 vs. 7.50, p < 0.001), and number of pups per female (13.00 vs. 40.67, p < 0.001) compared to the Mfffl/fl mice. The numbers of GV oocytes (29.37 vs. 39.10, p < 0.05), ovulated oocytes (9.81 vs. 20.91, p < 0.001) and 2-cell embryos (3.50 vs. 10.17, p < 0.05) were significantly lower in the Mfffl/fl; Gdf9-Cre mice. The blastocyst (32.32% vs. 68.44%, p < 0.05) embryo development rate per 2-cell embryos was also significantly lower. In vitro maturation revealed that of Mfffl/fl; Gdf9-Cre GV oocytes had significantly lower rates of GV break-down (68.89% vs. 91.57%, p < 0.05) and polar body extrusion (42.76% vs. 81.87%, p < 0.05). The ratios of aberrant spindle (71.56% vs. 23.74%, p < 0.001) and misaligned chromosomes (71.82% vs. 23.14%, p < 0.01) in ovulated oocytes from Mfffl/fl; Gdf9-Cre mice were significantly increased. In terms of mitochondrial dynamics, the mitochondrial size (0.59 µm2 vs. 0.15µm2, p < 0.001) and the ratio (62.50% vs. 18.67%, p < 0.01) of abnormal mitochondrial distribution (unevenly aggregated) in Mfffl/fl; Gdf9-Cre oocytes significantly elevated. This study found that the Mff played a crucial role in oocyte maturation and early embryonic development. Limitations, reasons for caution Further research is needed to determine the downstream targets or pathways of Mff in oocytes. Wider implications of the findings These results shed light on the important role played by MFF in ensuring proper mitochondrial dynamics in oocytes and underscore the significance of this protein in female fertility. This study provides insights into potential therapeutic targets for infertility and related diseases in humans. Trial registration number not applicable

Gonadotropin elevation is ootoxic to ovulatory oocytes and inhibits oocyte maturation, and activin decoy receptor ActRIIB:Fc therapeutically restores maturation

BackgroundElevated FSH often occurs in women of advanced maternal age (AMA, age ≥ 35) and in infertility patients undergoing controlled ovarian stimulation (COS). There is controversy on whether high endogenous FSH contributes to infertility and whether high exogenous FSH adversely impacts patient pregnancy rates.MethodsThe senescence-accelerated mouse-prone-8 (SAMP8) model of female reproductive aging was employed to assess the separate impacts of age and high FSH activity on the percentages (%) of viable and mature ovulated oocytes recovered after gonadotropin treatment. Young and midlife mice were treated with the FSH analog equine chorionic gonadotropin (eCG) to model both endogenous FSH elevation and exogenous FSH elevation. Previously we showed the activin inhibitor ActRIIB:Fc increases oocyte quality by preventing chromosome and spindle misalignments. Therefore, ActRIIB:Fc treatment was performed in an effort to increase % oocyte viability and % oocyte maturation.ResultsThe high FSH activity of eCG is ootoxic to ovulatory oocytes, with greater decreases in % viable oocytes in midlife than young mice. High FSH activity of eCG potently inhibits oocyte maturation, decreasing the % of mature oocytes to similar degrees in young and midlife mice. ActRIIB:Fc treatment does not prevent eCG ootoxicity, but it restores most oocyte maturation impeded by eCG.ConclusionsFSH ootoxicity to ovulatory oocytes and FSH maturation inhibition pose a paradox given the well-known pro-growth and pro-maturation activities of FSH in the earlier stages of oocyte growth. We propose the FOOT Hypothesis (“FSH OoToxicity Hypothesis), that FSH ootoxicity to ovulatory oocytes comprises a new driver of infertility and low pregnancy success rates in DOR women attempting spontaneous pregnancy and in COS/IUI patients, especially AMA women. We speculate that endogenous FSH elevation also contributes to reduced fecundity in these DOR and COS/IUI patients.Restoration of oocyte maturation by ActRIB:Fc suggests that activin suppresses oocyte maturation in vivo. This contrasts with prior studies showing activin A promotes oocyte maturation in vitro. Improved oocyte maturation with agents that decrease endogenous activin activity with high specificity may have therapeutic benefit for COS/IVF patients, COS/IUI patients, and DOR patients attempting spontaneous pregnancies.

Impairment of oocyte maturation as a mechanism of decreased fecundity in Japanese medaka (Oryzias latipes) exposed to the brominated flame retardant, 1,2,5,6-tetrabromocyclooctane (TBCO)

Inhibition of oocyte maturation is an understudied mechanism by which chemical stressors can impair fecundity of female fishes. The primary objective of the present study was to develop an assay to assess oocyte maturation disruption by chemical stressors in Japanese medaka (Oryzias latipes). First, an in vitro assay to assess maturation inducing hormone (MIH)-stimulated oocyte maturation in zebrafish was validated for use with Japanese medaka. Next, using the brominated flame retardant, 1,2,5,6-tetrabromocyclooctane (TBCO), which previously was shown to decrease fecundity of Japanese medaka and inhibit oocyte maturation in zebrafish, effects on oocyte maturation were quantified using in vitro and in vivo exposure. Adaptation of the protocol for in vitro MIH-stimulated maturation of stage IV oocytes from zebrafish was successful in inducing greater than 80% of stage IX oocytes from female Japanese medaka to mature. To assess effects of in vitro exposure, stage IX oocytes were exposed to 0, 2, 20, and 200 μg/L of TBCO, followed by exposure to MIH. The in vitro exposure caused a significant decrease in maturation of oocytes exposed to 20 and 200 μg/L of TBCO. To assess effects of TBCO on fecundity and oocyte maturation following in vivo exposure, sexually mature fish were fed a control, 100 μg/g, or 1000 μg/g concentration of TBCO-spiked fish food for 21 days, where fecundity was measured daily, and following the exposure, stage IX oocytes were excised to assess MIH-stimulated maturation. Fecundity and oocyte maturation were significantly decreased at either concentration of TBCO. Plasma concentrations of 17β-estradiol (E2) and hepatic abundances of transcripts of vitellogenin (vtgI and vtgII) were quantified, but there were no significant differences between treatments. Results suggest that inhibition of oocyte maturation is a mechanism by which TBCO decreases fecundity, and that in vitro assays of oocyte maturation might be predictive of fecundity in this species.

Associations between Urinary Concentrations of Disinfection Byproducts and in Vitro Fertilization Outcomes: A Prospective Cohort Study in China.

Experimental studies show that disinfection byproducts (DBPs) can inhibit oocyte maturation, decrease fertilization capacity, and impair embryo development, but human evidence is lacking. We aimed to evaluate the associations between exposure to drinking water DBPs and in vitro fertilization (IVF) outcomes. The study included 1,048 women undergoing assisted reproductive technology (ART) treatment between December 2018 and January 2020 from a prospective cohort study, the Tongji Reproductive and Environmental study in Wuhan, China. Exposure to DBPs was assessed by dichloroacetic acid (DCAA) and trichloroacetic acid (TCAA) in up to four urine samples, which were collected on the day of both enrollment and oocyte retrieval. Multivariable generalized linear mixed models, accounting for multiple IVF cycles per woman, were applied to evaluate the associations between urinary biomarkers of DBP exposures and IVF outcomes. Stratified analyses were used to explore the potential effect modifiers. The included 1,048 women underwent 1,136 IVF cycles, with 960 (91.6%), 84 (8.0%), and 4 (0.4%) women contributing one cycle, two cycles, and three cycles, respectively. We found that elevated quartiles of urinary DCAA and TCAA concentrations were associated with reduced numbers of total oocytes and metaphase II oocytes and that urinary DCAA concentrations with a lower proportion of best-quality embryos (all p for ). Moreover, elevated quartiles of urinary DCAA concentrations were associated with decreased proportions of successful implantation, clinical pregnancy, and live birth (14%, 15%, and 15% decreases in adjusted means comparing the extreme quartiles, respectively; all p for ). Stratification analyses showed that the inverse associations of urinary TCAA concentrations with multiple IVF outcomes were stronger among women y of age (p for ). Exposure to drinking water DBPs was inversely associated with some IVF outcomes among women undergoing ART treatment. Further study is necessary to confirm our findings. https://doi.org/10.1289/EHP12447.

Hybrid sterility in hermaphroditic Argopecten scallops: Mutated mitochondrial genes and abnormal expression of nuclear genes

Bay scallops (Argopecten irradians) are a commercially important bivalve species for fisheries and aquaculture in China, the USA, Peru and Chile, but their small size and serious inbreeding depression restrict the sustainable development of their aquaculture. We successfully hybridized bay scallops with Peruvian scallops (Argopecten purpuratus), and the F1 hybrids showed significant heterosis in growth, but the majority of them were sterile. Nevertheless, Argopecten scallops are typical hermaphroditic animals, resulting in a low proportion of interspecific hybrids. Hybrid sterility restricts the utilization of Argopecten scallop resources and causes reproductive isolation in speciation. However, the underlying mechanism of hybrid sterility remains unexplored in hermaphroditic scallops. In this study, we investigated the mechanism of hybrid sterility in hermaphroditic Argopecten scallops by examining mutations in mitochondrial genes and the expression of nuclear genes. Our results showed that the ATP content in the gonads of F1 hybrid scallops was significantly lower than that in their parents, indicating that energy deficiency may be an immediate cause of sterility in F1 hybrid scallops. Thirteen SNPs were detected in five mitochondrial genes of F1 hybrids, and 92.3% of which were transitions that could change the hydrophobicity of amino acids and protein structures, and the allele frequencies of the SNP loci were significantly different between sterile hybrids and fertile scallops. Abnormal interaction between the mitochondrial and nuclear genes might contribute to hybrid sterility through the following pathways: (1) inhibition of oocyte maturation by enhanced expression of Mfn2; (2) cell cycle arrest in G1 phase of oocytes by inhibited expression of CDK2; (3) cell apoptosis induced by mitochondrial apoptosis; (4) insufficient energy supply from abnormal mitochondria, and (5) mitochondrial dysfunction resulting from abnormal expression of other nuclear genes. The present results provide more information for understanding the mechanism of hybrid sterility in hermaphroditic animals and benefit extensive hybrid breeding.

P-650 Follicular extracellular vesicles of older women inhibit oocyte maturation

Abstract Study question Whether follicular extracellular vesicles of older women interfere with the quality of oocytes. Summary answer OLD-EVs induced the oxidative stress in the oocytes and inhibited oocyte maturation by increasing the abnormal mitochondria distribution and abnormal spindle rates. What is known already Ovary is a critical regulator of female fertility and the source of oocytes. With the modern tendency of delaying childbearing, ovarian aging has become an age-related disease. Ovarian aging is generally characterized by a gradual decrease in both the quantity and quality of oocytes. The pregnancy outcomes of older women were far from satisfying with higher abortion rates, birth defects rates, and lower live birth rates even after the assistance of IVF. However, the detailed mechanisms of decreased oocyte quality of aging women have not been clarified. Study design, size, duration Extracellular vesicles (EVs) were isolated from follicular fluid of older women (aged 40-50, n = 7, OLD-EVs) and younger women (aged 25-30, n = 6, YOUNG-EVs) via ultracentrifugation. Germinal vesicle (GV) oocytes from female ICR mice were co-cultured with OLD-EVs, YOUNG-EVs, or PBS (blank control), respectively. GV breakdown (GVBD) rate and maturation rate were calculated at two-hour and fourteen-hour of co-culture, respectively. Besides, oocyte mitochondria distribution, meiosis spindle morphology, and oxidative status were assessed in different groups. Participants/materials, setting, methods EVs were determined by western blotting, nanoparticle tracking analysis, and transmission electron microscopy. Fluorescence labeled EVs were used to visualize internalization by oocytes. Oocytes mitochondria and mitosis spindles were stained with fluorescence, and abnormal mitochondria rate or abnormal spindle rate was calculated. Reactive oxygen species (ROS) level was detected in the differently treated oocytes. Moreover, the expression of CAT, GSS, and SOD was determined in the oocytes using quantitative reverse transcription polymerase chain reaction. Main results and the role of chance Both OLD-EVs and YOUNG-EVs are bilayered vesicles, ranging from 100 to 150 nm and enriched in Alix, TSG101, and CD9. EVs could be internalized by oocytes within one hour. After coculture, GVBD rate was similar among the three groups; whereas maturation rate was significantly decreased in the OLD-EV group compared with YOUNG-EV group or PBS group. In addition, the abnormal mitochondria distribution rate or abnormal spindle rate were significantly increased in the OLD-EV group compared with PBS or YOUNG-EV group. The ROS level was increased in the PCOS-EV group compared with YOUNG-EV group, and the expression of CAT, GSS, and SOD was increased in the OLD-EV-treated oocytes. Limitations, reasons for caution Our study did not identify the contents of OLD-EVs and YOUNG-EVs, and the molecular mechanisms of dysregulations induced by OLD-EVs need further researches to investigate. Wider implications of the findings This work confirmed that EV-conducted cellular communication played an important role in oocyte maturation. The dysregulation of oocytes induced by OLD-EVs might be related to the poor oocyte quality of aging women, which provide a novel target to improve pregnancy outcomes of these patients. Trial registration number not applicable

Cyclic adenosine 3’5’monophosphate (cAMP) regulates meiotic maturation in goldfish oocytes Carassius auratus (Teleostei: Cyprinidae)

A good understanding of the mechanism of oocyte development and maturation, and the regulatory factors involved therein are important for the success of a breeding program especially of species used in aquaculture. It is crucial in a breeding program for females to supply high quality mature oocytes, which is generally achieved by fine-tuning oocyte meiotic arrest and resumption progression. Therefore, knowledge of the oocyte maturation process is fundamental for the development of methodologies to increase the success of fish production in aquaculture. TheFigure regulations of oocyte maturation still are not totally understood. The hypothesis whether elevated levels or a drop in intraoocyte cAMP triggers resumption of meiotic maturation was investigated in denuded goldfish oocytes. In the presence of agents that elevate cAMP like forskolin, dibutyryl cAMP, isobutyl methylxanthine, maturation of denuded oocytes was inhibited. Whereas 2’deoxyadenosine-3-monophosphate, an inhibitor of adenylate cyclase promoted oocyte maturation. Forskolin decreased spontaneous maturation as well as hormone-induced maturation of goldfish oocytes. At 10 µM forskolin added up to 4 h after hormonal stimulation completely inhibited oocyte maturation. Beyond 4 h, however, when oocyte maturation had already started, forskolin can no longer inhibit maturation. Intraoocyte cAMP assayed in denuded oocytes with or without hormonal stimulation decreased immediately within 30 minutes. With forskolin, it did not cause a significant decrease in cAMP not until at 4 h. The results were consistent with the findings that elevated levels of cAMP maintain meiotic arrest while a decrease in cAMP is necessary and sufficient to trigger resumption of meiotic maturation.

Inhibition of Oocyte Maturation by Follicular Extracellular Vesicles of Nonhyperandrogenic PCOS Patients Requiring IVF.

Polycystic ovary syndrome (PCOS) is one of the most common diseases that contribute to subfertility. Recent evidence showed that oocytes of women with PCOS matured in vitro away from the follicular fluid presented better potentials, whereas the reason remained unclear. This work aimed to investigate whether follicular extracellular vesicles (EVs) of PCOS patients interfere with the quality of oocytes. Follicular EVs of women with PCOS (PCOS-EVs) and control women (CTRL-EVs) were isolated and determined using Western blotting, nanoparticle tracking analysis, and transmission electron microscopy. The 2 types of EVs were co-cultured with murine germinal vesicle oocytes, respectively. Fluorescence-labeled EVs were used to visualize internalization by oocytes. After co-culture, oocyte maturation rates were calculated. Mitochondria distribution and reactive oxygen species (ROS) level were detected in the different groups. Spindle morphology was evaluated using immunofluorescence. Moreover, the expression of catalase (CAT), glutathione synthetase (GSS), and superoxide dismutase (SOD) was determined in the oocytes. Both PCOS-EVs and CTRL-EVs are bilayered vesicles, approximately 100 to 150 nm in size, and enriched in EV-associating protein markers. EVs were internalized by oocytes within 1 hour. Oocyte maturation rate decreased significantly in the PCOS-EV group compared with the CTRL-EV group, whereas the abnormal mitochondria distribution rate and abnormal spindle rate were significantly increased in the PCOS-EV group. Moreover, PCOS-EVs increased the ROS level and the expression of CAT, GSS, and SOD in the oocytes. PCOS-EVs interfered with oocyte mitochondria and spindles and inhibited oocyte maturation. Moreover, oxidative stress induced by PCOS-EVs might be a potential cause.

Environmentally relevant concentrations of selenite trigger reproductive toxicity by affecting oocyte development and promoting larval apoptosis

As a trace element, selenium (Se) has been widely added to food to maintain the physiological homeostasis of the organism. The adverse effects of Se on the reproduction of zebrafish have been investigated, however, the effects of Se on the maturation and apoptosis of zebrafish oocytes remain unclear. In this study, zebrafish embryos (2 h post fertilization, hpf) were exposed to 0, 12.5, 25, 50, and 100 μg Se/L for 120 days. The results demonstrated that exposure to selenite decreased the gonad-somatic index (GSI) and cumulative production of eggs, inhibited oocyte maturation (OM), and increased oocyte apoptosis in females. Exposure to selenite decreased the contents of sex hormones (E2) in the serum and increased the levels of reactive oxygen species (ROS) and cyclic adenosine monophosphate (cAMP) in the ovary. Furthermore, exposure to selenite downregulated the transcription level of genes on the HPG axis, decreased the phosphorylation level of CyclinB and the protein content of cAMP-dependent protein kinase (Pka), and upregulated the expression of genes (eif2s1a and chop) and proteins (Grp78, Chop) related to endoplasmic reticulum stress (ERS) and apoptosis. Moreover, maternal exposure to selenite resulted in the apoptosis of offspring and upregulated the content of ROS and the transcription level of genes related to ERS and apoptosis.

Inhibition of Oocyte Maturation in the Estuarine Crab Neohelice Granulata, by the Effect of Anti-Inflammatory Drugs.

The drugs ibuprofen and diclofenac were assessed in vivo on adult females of the estuarine crab Neohelice granulata. In a first, preliminary assay comprising 60-d, a significant (p < 0.05) lower content of total vitellogenic proteins was detected in the ovary at 10mg/L of each drug. In a second 90-d assay, comprising the exposure of crabs to 5mg/L of each drug during the entire pre-reproductive period, a significant (p < 0.05) decrease in the proportion of vitellogenic oocytes was observed by effect of diclofenac. The same effect was also observed in a third assay only comprising the last month of the pre-reproductive period, at 5mg/L of diclofenac, and also at a mixture of both drugs; besides, this mixture significantly (p < 0.05) increased the proportion of reabsorbed vitellogenic oocytes. The obtained results indicate that the effect of diclofenac is critical at the final stage of ovarian maturation, when the participation of prostaglandins is relevant.

SET improved oocyte maturation by serine/threonine protein phosphatase 2A and inhibited oocyte apoptosis in mouse oocytes

SET is a multifunctional protein involved in a variety of molecular processes such as cell apoptosis and cell-cycle regulation. In ovaries SET is predominantly expressed in theca cells and oocytes. In polycystic ovary syndrome (PCOS) patients the expression of SET was increased than healthy people. The current study was designed to determine whether SET plays a role in oocyte maturation and apoptosis, which may provide clues for the underlying pathological mechanism of follicular development in PCOS patients. Oocytes at germinal vesicle (GV) stage were collected from 6-week-old female ICR mice ovaries. The expression of SET was manipulated by AdCMV-SET and AdH1-SiRNA/SET adenoviruses. SET overexpression improved oocyte maturation whereas SET knockdown inhibited oocyte maturation. Moreover, SET negatively regulated serine/threonine protein phosphatase 2A (PP2A) activity in oocytes. Treatment with PP2A inhibitor okadaic acid (OA) promoted oocyte maturation. Furthermore, PP2A knockdown confirmed the role of PP2A in oocyte maturation, and OA was able to block the AdH1-SiRNA/SET-mediated inhibition on oocyte maturation. The central role of PP2A in SET-mediated regulation of oocyte maturation was confirmed by the finding that SET increased the expression of bone morphogenetic protein 15 (BMP15) and growth differentiation factor 9 (GDF9) and PP2A inhibited their expressions. Besides, SET inhibited oocyte apoptosis through decreasing the expression of caspase 3 and caspases 8, while PP2A had no effect on oocyte apoptosis. SET promoted oocyte maturation by inhibiting PP2A activity and inhibited oocyte apoptosis in mouse in-vitro cultured oocytes, which may provide a pathologic pathway leading to impaired oocyte developmental competence in PCOS.

The Molecular Quality and Mitochondrial Activity of Porcine Cumulus–Oocyte Complexes Are Affected by Their Exposure to Three Endocrine-Active Compounds under 3D In Vitro Maturation Conditions

Thus far, the potential short- and long-term detrimental effects of a variety of environmental chemicals designated as endocrine-active compounds (EACs) have been found to interfere with histo- and anatomo-physiological functions of the reproductive system in humans and wildlife species. For those reasons, this study sought to examine whether selected EACs, which encompass the fungicide vinclozolin (Vnz), the androgenic anabolic steroid nandrolone (Ndn) and the immunosuppressant cyclosporin A (CsA), affect the developmental competence and molecular quality (MQ) of porcine cumulus–oocyte complexes (COCs) subjected to in vitro maturation (IVM) under 3D culture conditions. The COCs underwent 3D-IVM in the presence of Vnz, Ndn or CsA for 48 h. To explore whether the selected EACs induce internucleosomal DNA fragmentation in cumulus cells (CCs), TUNEL-assisted detection of late apoptotic cells was performed. Additionally, for the detailed evaluation of pro- and antiapoptotic pathways in COCs, apoptosis proteome profiler arrays were used. To determine changes in intracellular metabolism in COCs, comprehensive assessments of mitochondrial ultrastructure and activity were carried out. Moreover, the relative abundances (RAs) of mRNAs transcribed from genes that are involved in scavenging reactive oxygen species (ROS), such as SIRT3 and FOXO3, and intramitochondrial bioenergetic balance, such as ATP synthase subunit (ATP5A1), were ascertained. Finally, to investigate the extent of progression of oocyte maturation, the intraooplasmic levels of cAMP and the RAs of mRNA transcripts encoding regulatory and biocatalytic subunits of a heterodimeric meiosis-promoting factor, termed cyclin B1 (CCNB1) and cyclin-dependent kinase 1 (CDC2), were also estimated. The obtained results provide, for the first time, strong evidence that both Vnz and Ndn decrease the developmental competence of oocytes and stimulate apoptosis processes in CCs. The present study is also the first to highlight that Vnz accelerates the maturation process in immature oocytes due to both increased ROS production and the augmented RA of the CCNB1 gene. Furthermore, Vnz was proven to trigger proapoptotic events in CCs by prompting the activity of the FOXO3 transcription factor, which regulates the mitochondrial apoptosis pathway. In turn, Ndn was shown to inhibit oocyte maturation by inducing molecular events that ultimately lead to an increase in the intraooplasmic cAMP concentration. However, due to the simultaneous enhancement of the expression of TNF-β and HSP27 proteins in CCs, Ndn might be responsible for the onset of their neoplastic transformation. Finally, our current investigation is the first to clearly demonstrate that although CsA did not interfere with the nuclear and cytoplasmic maturation of oocytes, by inducing mitophagy in CCs, it disrupted oocyte metabolism, consequently attenuating the parameters related to the MQ of COCs. Summing up, Vnz, Ndn and CsA reduced not only the processes of growth and IVM but also the MQ of porcine COCs, which might make them unsuitable for assisted reproductive technologies (ARTs) such as in vitro fertilization by either gamete co-incubation or intracytoplasmic sperm injection (ICSI) and cloning by somatic cell nuclear transfer (SCNT).

Inhibition of Oocyte Maturation by Malathion and Structurally Related Chemicals in Zebrafish (Danio rerio) After In Vitro and In Vivo Exposure.

Oogenesis is the process by which a primary oocyte develops into a fertilizable oocyte, making it critical to successful reproduction in fish. In zebrafish (Danio rerio), there are five stages of oogenesis. During the final step (oocyte maturation), the maturation-inducing hormone 17α,20β-dihydroxy-4-pregnen-3-one (MIH) activates the membrane progestin receptor, inducing germinal vesicle breakdown. Using in vitro assays, it has been shown that anthropogenic stressors can dysregulate MIH-induced oocyte maturation. However, it is unknown whether the in vitro assay is predictive of reproductive performance after in vivo exposure. We demonstrate that a known inhibitor of oocyte maturation, malathion, and a structurally related chemical, dimethoate, inhibit oocyte maturation. However, malaoxon and omethoate, which are metabolites of malathion and dimethoate, did not inhibit oocyte maturation. Malathion and dimethoate inhibited maturation to a similar magnitude when oocytes were exposed for 4 h in vitro or 10 days in vivo, suggesting that the in vitro zebrafish oocyte maturation assay might be predictive of alterations to reproductive performance. However, when adult zebrafish were exposed to malathion for 21 days, there was no alteration in fecundity or fertility in comparison with control fish. Our study supports the oocyte maturation assay as being predictive of the success of in vitro oocyte maturation after in vivo exposure, but it remains unclear whether inhibition of MIH-induced oocyte maturation in vitro correlates to decreases in reproductive performance. Environ Toxicol Chem 2022;41:1381-1389. © 2022 SETAC.

Exposure of mouse oocytes to N,N-dimethylformamide impairs mitochondrial functions and reduces oocyte quality.

N,N-dimethylformamide (DMF) is a widely-used solvent for the synthesis of synthetic fibers such as polyacrylonitrile fiber, and can also be used to make medicine. Although this organic solvent has multipurpose applications, its biological toxicity cannot be ignored and its impact on mammalian reproduction remains largely unexplored. Our study found that DMF exposure inhibited oocyte maturation and fertilization ability. Transcriptomic analysis indicated that DMF exposure changed the expression of genes and transposable elements in oocytes. Subcellular structure examination found that DMF exposure caused mitochondrial dysfunction, abnormal aggregation of mitochondria and decreased mitochondrial membrane potential in mouse oocytes. Its exposure also caused abnormal distribution of Golgi apparatus and endoplasmic reticulum which formed large number of clusters. In addition, oxidative stress occurs in oocytes exposed to DMF, which was manifested by an increase in the level of reactive oxygen species. We found that DMF exposure induced disordered spindle and chromosomes abnormality. Meanwhile, we examined various histone modification levels in oocytes exposed to DMF and found that DMF exposure reduced H3K9me3, H3K9ac, H3K27ac, and H4K16ac levels in mouse oocytes. Moreover, DMF-treated oocytes failed to form pronuclei after fusion with normal sperm. Collectively, DMF exposure caused mitochondrial damage, oxidative stress, spindle assembly and chromosome arrangement disorder, leading to oocyte maturation arrest and fertilization failure.

Inhibition of oocyte maturation by nitric oxide synthase 1 (NOS1) in zebrafish

It is well-documented that nitric oxide (NO) is an important regulator of oocyte maturation in mammals. Conversely, the function of NO during oocyte maturation has received little attention in nonmammalian vertebrates. NO is produced from L-arginine through the action of the enzyme NO synthase (NOS). Herein, we examined the expression, hormonal regulation, and involvement of NOS in meiotic signaling in zebrafish oocyte maturation. Three types of nos genes, nos1, nos2a, and nos2b, have been identified in zebrafish. We found that the expression of nos1 was highest in the ovary among the three nos genes, with maximal expression in full-grown (FG)-stage follicles during folliculogenesis. In addition, the concentration of NO was reduced during oocyte maturation and this corresponded with the decreased expression of nos1 in the follicular cell layers, suggesting that NOS1-derived NO may be one of the inhibitors of oocyte maturation in zebrafish. This is the first description of nos1 involvement in oocyte maturation in vertebrates. Moreover, the NO donor SNAP (S-nitroso-l-acetyl penicillamine) partially attenuates human chorionic gonadotropin (hCG)- and 17,20β-P-induced GVBD (germinal vesicle breakdown), perhaps by increasing cGMP levels during oocyte maturation. Finally, our results showed that SNAP and the cGMP analog 8-Br-cGMP inhibited hCG-induced mitogen-activated protein kinase (MAPK) activation, further indicating that NO and cGMP block oocyte maturation in zebrafish.

Replication protein A is required for juvenile hormone-dependent vitellogenesis and oocyte maturation in locusts

Aside from inhibiting insect metamorphosis, juvenile hormone (JH) has a well-known role in stimulating various aspects of insect reproduction. Replication protein A (RPA), a heterotrimeric complex comprised of RPA1, RPA2 and RPA3 subunits plays an essential role in DNA replication and DNA repair. Here we report that RPAs are highly expressed in the fat body of adult female locust, Locusta migratoria. While RPA1 is upregulated by the JH receptor Methoprene-tolerant (Met), RPA2 and RPA3 expression appears to be primarily controlled by Forkhead box O transcription factor (FoxO). Knockdown of RPA1, RPA2 or RPA3 results in markedly reducd vitellogenin (Vg) expression in the fat body, accompanied by arrested ovarian growth and inhibited oocyte maturation. In addition, depletion of an RPA subunit leads to increased expression of other RPA subunits as well as a pro-apoptotic gene, Smac that is involved in DNA repair and apoptosis. The data indicate a crucial role of RPAs in JH-dependent vitellogenesis and oocyte maturation.

Toxic effects of patulin on mouse oocytes and its possible mechanisms

Patulin is a secondary metabolite mainly secreted by fungi and is the most common mycotoxin found in apples and apple-based products. For the past few years, numerous studies suggested the wide distribution and toxicity of patulin. In this study, we investigated the toxic effect of patulin on mouse oocytes and its possible mechanisms. The results showed that patulin treatment did not affect meiotic resumption, but inhibited oocyte maturation as indicated by failure of first polar body extrusion. Further mechanistic study showed that patulin treatment disturbed normal spindle assembly, chromosome alignment and morphology. We also found increased oxidative stress by testing the level of ROS and decreased mitochondrial membrane potential, indicating mitochondria dysfunction. In summary, our results suggest that patulin treatment causes oocyte meiotic arrest by disturbing normal spindle assembly and chromosome alignment, which may be caused by dysfunctions of mitochondria.

P–129 Follicular extracellular vesicles of women with polycystic ovarian syndrome inhibit oocyte maturation

Abstract Study question Does follicular extracellular vesicles of women with polycystic ovarian syndrome (PCOS-EVs) interfere with the quality of oocytes? Summary answer PCOS-EVs induced oxidative stress in the oocytes and inhibited oocyte maturation by increasing the abnormal mitochondria distribution and abnormal spindle rates. What is known already Polycystic ovarian syndrome (PCOS) is a common endocrine disorder in women of reproductive age, with a prevalence up to 10%. Women with PCOS are characterized by a clustering of features, including hyperandrogenism, polycystic ovarian morphology, and notably, anovulation. Although international guidelines recommend assisted reproduction techniques to be an effective resort for PCOS patients to conceive. However, even after overcoming ovulatory dysfunction via ovulation induction, the pregnancy outcomes of patients with PCOS were far from satisfying with lower fertilization, cleavage, and implantation rates, implicating that theoocyte quality of these patients are affected. Whereas the mechanisms have not been elucidated yet. Study design, size, duration Follicular fluid of PCOS patients (n = 10) and healthy controls (n = 10) were collected and used for extracellular vesicles (EVs) isolation via ultracentrifugation. Germinal vesicle (GV) oocytes collected from female ICR mice were cocultured with RIF-EVs or FER-EVs, respectively, and PBS served as a blank control. GV breakdown (GVBD) rate and maturation rate were calculated at two-hour and fourteen-hour of co-culture, respectively. Besides, oocyte mitochondria distribution, meiosis spindle morphology, and oxidative status were assessed in different groups. Participants/materials, setting, methods EVs were determined by western blotting, nanoparticle tracking analysis, and transmission electron microscopy. Fluorescence labeled EVs were used to visualize internalization by oocytes. Oocytes mitochondria and mitosis spindles were stained with fluorescence, and abnormal mitochondria rate or abnormal spindle rate was calculated. Reactive oxygen species (ROS) level was detected in the differently treated oocytes. Moreover, the expression of CAT, GSS, and SOD was determined in the oocytes using quantitative reverse transcription polymerase chain reaction. Main results and the role of chance Both PCOS-EVs and CTRL-EVs are bilayered vesicles, ranging from 100 to 150 nm, and enriched in Alix, TSG101, and CD9. EVs could be internalized by oocytes within one hour. After coculture, the GVBD rate was similar among the three groups; whereas the maturation rate was significantly decreased in the PCOS-EV group compared with CTRL-EV group or PBS group. In addition, the abnormal mitochondria distribution rate or abnormal spindle rate were significantly increased in the PCOS-EV group compared with PBS or CTRL-EV group. The ROS level was increased in the PCOS-EV group compared with CTRL-EV group, and the expression of CAT, GSS, and SOD was increased in the PCOS-EV-treated oocytes. Limitations, reasons for caution Our study did not identify the contents of PCOS-EVs and CTRL-EVs, and the molecular mechanisms of dysregulations induced by PCOS-EVs need further researches to investigate. Wider implications of the findings: This work confirmed that EV-conducted cellular communication played an important role in oocyte development in women with PCOS. The dysregulation of oocytes induced by PCOS-EVs might be related to the poor oocyte quality of women with PCOS, which may provide a novel target to improve pregnancy outcomes of these patients. Trial registration number Not applicable

Effect of perfluorohexane sulfonate on pig oocyte maturation, gap-junctional intercellular communication, mitochondrial membrane potential and DNA damage in cumulus cells in vitro

Perfluorohexane sulfonate (PFHxS) is one of the most abundant perfluorinated compounds in the environment. Exposure to this compound has been correlated to a decrease in human fertility, although the molecular and cellular mechanisms underlying this correlation have not been described. The adverse reproductive effects of PFHxS could be based on alterations in oocyte maturation, the process rendering oocytes competent for fertilization. The aim of this study was to evaluate the effect of PFHxS on porcine oocyte viability and maturation in vitro, as well as on gap-junctional intercellular communication (GJIC) in cumulus–oocyte complexes (COCs), oocyte mitochondrial membrane potential (mΔΨ) and DNA damage in cumulus cells, as possible mechanisms of action. PFHxS caused cytotoxicity (medium lethal concentration, LC50 = 329.1 μM) and inhibition of oocyte maturation (medium inhibitory concentration, MIC50 = 91.68 μM). GJIC was not affected in exposed COCs. However, the mitochondrial membrane potential was significantly decreased in PFHxS-exposed oocytes at the germinal vesicle breakdown (GVBD) stage. In addition, exposure to PFHxS induced DNA damage in cumulus cells. Thus, inhibition of oocyte maturation by PFHxS could be attributed to a decreased oocyte mΔΨ at the GVBD and to DNA damage of the cumulus cells that support the oocyte.

Actinomycin D causes oocyte maturation failure by inhibiting chromosome separation and spindle assembly†.

Actinomycin D (ActD) has been considered as one of the most effective and safe chemotherapeutic medications for treating a number of cancers. Although ActD has been used in the treatment of gynecological tumors and pediatric tumors for more than 50years, the toxic effects of ActD on mammalian oocytes remain unknown. In this study, the influence of ActD on mouse and human oocyte maturation and the possible mechanisms were investigated. Notably, ActD inhibited oocyte maturation and arrested oocytes at the metaphase I (MI) stage in a dose-dependent manner. In addition, ActD arrested oocyte maturation when the oocytes were treated at different successive stages, including the germinal vesicle (GV), germinal vesicle breakdown, and MI stages. In ActD-treated oocytes, disordered chromosome condensation and irregular spindle assembly occurred, resulting in incomplete chromosome segregation and oocytes arresting at the MI phase; these results possibly occurred because ActD triggered the formation of reactive oxygen species, resulting in DNA damage and decreased ATP in mouse GV oocytes. Besides, in vivo treatment with ActD also inhibited mouse oocyte maturation. Similar effects were seen in human oocytes. Collectively, our results indicated that ActD exposure disrupted oocyte maturation by increasing DNA damage, which is a finding that might help with optimizing future methods for female fertility preservation before undergoing chemotherapy.